Influenza viruses belong to the family of Orthomyxoviridae which contains viruses with negative sense, single-stranded and segmented RNA genomes. There are five different genera under this family: influenza A, B, C, Thogotovirus and Isavirus. Within the first three genera, influenza A and C infect humans, swine, birds and other mammals, while influenza B almost solely infects humans (Peter Palese, 2007). Influenza viruses are estimated to cause more than 5 million serious cases of illness annually worldwide. The symptoms of influenza infection range from asymptomatic to primary viral pneumonia which can be fatal. The clinical manifestations generally include headache, chills, dry cough, high fever, significant myalgias, malaise and anorexia. The clinical manifestations in children are similar to those in adults except they can exhibit high fever accompanied by febrile convulsions (Peter F Wright, 2007).
Influenza virions are highly pleomorphic and they occur in many shapes including ovoid and long filamentous particles. Structurally, each influenza virion consists of haemagglutinin (HA) (135 Å trimer) and neuraminidase (NA) (60-100 Å trimer) proteins which project prominently on the outer surface of the lipid envelope and present in a 4-5:1 ratio. The inner side of the lipid envelope is lined by the matrix protein, encapsulating a core of negative sense RNAs which consists of 8 segments (7 segments for Influenza C) and are wrapped helically by nucleoprotein. It has been reported that HA and NA play a vital role in viral infection. The HA is responsible for binding to the sialic acid receptors on the membrane of infected cells and viral entry by membrane fusion, whereas the NA is required during the release and spread of progeny virions, following the intracellular viral replication cycle (Peter Palese, 2007).
The RNA genome of influenza viruses can mutate, resulting in “antigenic drift’. In addition, two different subtypes can combine and form a new subtype when they co-infect the same host, also resulting in “antigenic shift”. These two types of mutations may cause slight or complete alteration on the surface protein type including its shape, infectious ability and pathogenicity; which eventually render an influenza virus a pandemic or epidemic strain (Peter F Wright, 2007). More than four major pandemics caused by influenza viruses have been reported since the beginning of the last century which resulted in great losses to the international economy and human productivity (Kilbourne, 2006). Recently, a swine origin influenza A (H1N1) virus spread fast around the globe and caused reasonably severe mortality and morbidity among the human population (Echevarria-Zuno et al., 2009). It has been suggested that a highly pathogenic epizootic H5N1 virus would be the next pandemic although it has been reported that it can only spread among bird populations with intermittent human infections at a very high rate for mortality (Webster and Govorkova, 2006). Recently, novel reassortant avian H7N9 viruses were found to be associated with severe and fatal respiratory disease among humans in China (Gao et al., 2013).
Previously, the transmission of avian viruses to humans was not considered as a serious threat. It had been reported that avian viruses do not replicate efficiently in experimentally infected humans (Beare A S, 1991). Consistent with that report, there was no report on serious outbreaks of highly pathogenic avian influenza viruses in humans until 1997 except a very few isolated avian-human transmission cases and they lacked the ability to transmit efficiently from person-to-person. However, recent evidence from person-to-person transmission of H5N1 viruses suggests an increase in the chances of human adapted avian viruses as potential pandemic candidates (Ungchusak et al., 2005; Yang et al., 2007).
Nevertheless, despite the extensive efforts made over the decades around the world to control the infection of influenza viruses, only two classes of anti-influenza virus drugs are currently available for effective treatment. One class acts as an antagonist to the influenza A M2 ion channel protein (amantadine and its derivative rimantadine) (Kolocouris et al., 2007) and the other, which includes oseltamivir, acts in influenza A and
B as a neuraminidase inhibitor (Zanamivir, Oseltamivir phosphate & Peramivir) (Kim et al., 1997; von Itzstein et al., 1993; Yun et al., 2008) which binds to the NA protein and makes the virus progeny unable to escape from the host cell and infect other cells. Unfortunately, these anti-viral drugs possess undesirable side effects. Moreover, most of the viral strains have now built resistance to the M2 ion channel blocker and to a lesser extent to the neuraminidase inhibitor, which leads to reduced drug efficacy and increased toxicity (McKimm-Breschkin, 2000; Whitley et al., 2013). It has also been reported that oseltamivir could acerbate the illness and sometimes even cause death (Hama et al., 2011). Various approaches targeting viral RNA transcription (Perez et al., 2010), virus-cell attachment (Jones et al., 2006), prevention of the proteolytic activation of HA (Zhirnov and Klenk, 2011), and prevention of virion budding (Stiver, 2003) have been developed, but so far none of the compounds have been successfully translated into an anti-viral drug for clinical use. Therefore, there is an urgent need to develop novel anti-influenza therapeutics having alternative modes of action.
In recent years, the pharmaceutical companies are facing continuous economic pressure due to the increased cost for R&D, toxicity, lack of efficacy, clinical safety and decreased number of approved new molecular entities. Kola and Landis reported that approximately 90% of these new molecular entities failed during drug development (Kola and Landis, 2004). Therefore, there is an urgent need to obtain alternative approaches to improve pharmaceutical R&D productivity. It is believed herein that peptide-based therapeutics could be one of the best options.
Peptide therapeutics may offer advantages over traditional small molecule based drugs. Firstly, they may offer great efficacy, specificity and selectivity as they often represent an active unit of a protein molecule and thus avoid substantial non-specific binding interactions (Hummel, Reineke, and Reimer, 2006). Secondly, as the degradation products of the peptides are amino acids, the chances of drug-drug interactions should be very much minimized and any toxicity related issues may be avoided (Loffet, 2002). Due to their smaller size than proteins and antibody based therapeutics, peptides can easily penetrate into tissues and organs (Ladner et al., 2004) and are generally less immunogenic (McGregor, 2008). Moreover, their cost of manufacturing can be lower than other bio-therapeutics.
A 9 amino acid peptide (C-P1) from a phage display library (New England Biolabs, USA) has been reported to possess anti-influenza activity in vitro and in ovo. See, for example, Rajik et al “Identification and characterization of a novel antiviral peptide against avian influenza virus H9N2, Virology Journal, (2009) 6:74, doi:10.1186/1743-422X-6-74”, Rajik et al “A novel peptide inhibits influenza virus replication by preventing the viral attachment to the host cells, International Journal of Biological Sciences, 2009; 5(6):543-548”, Malaysian patent application No. PI20082061, PCT patent application publication No. WO 2009/151313 A1, European patent No. EP2300492 B1, Japan patent application publication No. JP 2011522561A and U.S. Pat. No. 8,883,480. The peptide C-P1 reportedly inhibits the avian influenza A virus H9N2 replication with modest efficacy. There are however no reports regarding any activity of C-P1 against influenza viruses which infect humans. The precise mechanism of action has not been reported. It has been discovered herein that the activity of C-P1 may be insufficient for practical use, and therefore, there remains an unmet medical need for medicaments capable of preventing and/or treating influenza virus infection.